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The range of commercially available array platforms and analysis software packages is expanding and their utility is improving, making reliable detection of copy-number variants (CNVs) relatively straightforward. Reliable interpretation of CNV data, however, is often difficult and requires expertise. With our knowledge of the human genome growing rapidly, applications for array testing continuously broadening, and the resolution of CNV detection increasing, this leads to great complexity in interpreting what can be daunting data. Correct CNV interpretation and optimal use of the genotype information provided by single-nucleotide polymorphism probes on an array depends largely on knowledge present in various resources. In addition to the availability of host laboratories' own datasets and national registries, there are several public databases and Internet resources with genotype and phenotype information that can be used for array data interpretation. With so many resources now available, it is important to know which are fit-for-purpose in a diagnostic setting. We summarize the characteristics of the most commonly used Internet databases and resources, and propose a general data interpretation strategy that can be used for comparative hybridization, comparative intensity, and genotype-based array data., Competing Interests: Statement: The authors declare no conflict of interest.

The analytical resolution of individual chromosome peaks in the flow karyotype of cell lines is dependent on sample preparation and the detection sensitivity of the flow cytometer. We have investigated the effect of laser power on the resolution of chromosome peaks in cell lines with complex karyotypes. Chromosomes were prepared from a human gastric cancer cell line and a cell line from a patient with an abnormal phenotype using a modified polyamine isolation buffer. The stained chromosome suspensions were analyzed on a MoFlo sorter (Beckman Coulter) equipped with two water-cooled lasers (Coherent). A bivariate flow karyotype was obtained from each of the cell lines at various laser power settings and compared to a karyotype generated using laser power settings of 300 mW. The best separation of chromosome peaks was obtained with laser powers of 300 mW. This study demonstrates the requirement for high-laser powers for the accurate detection and purification of chromosomes, particularly from complex karyotypes, using a conventional flow cytometer., (Copyright 2010 International Society for Advancement of Cytometry.)

To elucidate the pathogenesis of hepatocellular carcinoma (HCC) and develop useful prognosis predictors, it is necessary to identify biologically relevant genomic alterations in HCC. In our study, we defined recurrently altered regions (RARs) common to many cases of HCCs, which may contain tumor-related genes, using whole-genome array-CGH and explored their associations with the clinicopathologic features. Gene set enrichment analysis was performed to investigate functional implication of RARs. On an average, 23.1% of the total probes were altered per case. Mean numbers of altered probes are significantly higher in high-grade, bigger and microvascular invasion (MVI) positive tumors. In total, 32 RARs (14 gains and 18 losses) were defined and 4 most frequent RARs are gains in 1q21.1-q32.1 (64.5%), 1q32.1-q44 (59.2%), 8q11.21-q24.3 (48.7%) and a loss in 17p13.3-p12 (51.3%). Through focusing on RARs, we identified genes and functional pathways likely to be involved in hepatocarcinogenesis. Among genes in the recurrently gained regions on 1q, expression of KIF14 and TPM3 was significantly increased, suggesting their oncogenic potential in HCC. Some RARs showed the significant associations with the clinical features. Especially, the recurrent loss in 9p24.2-p21.1 and gain in 8q11.21-q24.3 are associated with the high tumor grade and MVI, respectively. Functional analysis showed that cytokine receptor binding and defense response to virus pathways are significantly enriched in high grade-related RARs. Taken together, our results and the strategy of analysis will help to elucidate pathogenesis of HCC and to develop biomarkers for predicting behaviors of HCC., ((c) 2008 Wiley-Liss, Inc.)

Background: The analysis and isolation of high numbers of chromosomes smaller than 3 Mb in size (microchromosomes) with good purity is dependent primarily on the detection sensitivity of the flow cytometer and the precision of the sort unit. The aim of this study was to investigate the capability of using a conventional flow cytometer for the detection and sorting at high purity microchromosomes with an estimated size of 2.7 Mb., Methods: Chromosomes were isolated from a human cell line containing a pair of X-derived microchromosomes, using a modified polyamine isolation buffer. The chromosome preparation was labeled with Hoechst and Chromomycin and analyzed and purified using a MoFlo sorter (DAKO) configured for high-speed sorting. The purity of the flow-sorted microchromosomes was assessed by reverse chromosome painting., Results: Improved resolution of the peak of microchromosomes in a bivariate plot of Hoechst versus Chromomycin fluorescence was obtainable after discriminating clumps and debris based on gating data within a FSC versus pulse width plot., Conclusions: Chromosomes of smaller size, less than 3 Mb, can be detected with high resolution and flow-sorted with high purity using a conventional flow sorter.

The most frequent cause of familial clear cell renal cell carcinoma (RCC) is von Hippel-Lindau disease and the VHL tumor suppressor gene (TSG) is inactivated in most sporadic clear cell RCC. Although there is relatively little information on the mechanisms of tumorigenesis of clear cell RCC without VHL inactivation, a subset of familial cases harbors a balanced constitutional chromosome 3 translocation. To date nine different chromosome 3 translocations have been associated with familial or multicentric clear cell RCC; and in three cases chromosome 6 was also involved. To identify candidate genes for renal tumorigenesis we characterized a constitutional translocation, t(3;6)(q22;q16.1) associated with multicentric RCC without evidence of VHL target gene dysregulation. Analysis of breakpoint sequences revealed a 1.3-kb deletion on chromosome 6 within the intron of a 2 exon predicted gene (NT_007299.434). However, RT-PCR analysis failed to detect the expression of this gene in lymphoblast, fibroblast, or kidney tumor cell lines. No known genes were disrupted by the translocation breakpoints but several candidate TSGs (e.g., EPHB1, EPHA7, PPP2R3A RNF184, and STAG1) map within close proximity to the breakpoints., ((c) 2007 Wiley-Liss, Inc.)

Background: One of the major factors which influences the chromosome purity achievable particularly during high speed sorting is the analytical resolution of individual chromosome peaks in the flow karyotype, as well as the amount of debris and fragmented chromosomes. We have investigated the factors involved in the preparation of chromosome suspensions that influence karyotype resolution., Methods: Chromosomes were isolated from various human and animal cell types using a series of polyamine buffer isolation protocols modified with respect to pH, salt concentration, and chromosome staining time. Each preparation was analyzed on a MoFlo sorter (DAKO) configured for high speed sorting and the resolution of the flow karyotypes compared., Results: High resolution flow cytometric data was obtained with chromosomes optimally isolated using hypotonic solution buffered at pH 8.0 and polyamine isolation buffer (with NaCl excluded) between pH 7.50 and 8.0. Extending staining time to more than 8 h with chromosome suspensions isolated from cell lines subjected to sufficient metaphase arrest times gave the best result with the lowest percentage of debris generated, tighter chromosome peaks with overall lower coefficients of variation, and a 1- to 5-fold increase in the yield of isolated chromosomes., Conclusions: Optimization of buffer pH and the length of staining improved karyotype resolution particularly for larger chromosomes and reduced the presence of chromosome fragments (debris). However, the most interesting and surprising finding was that the exclusion of NaCl in PAB buffer improved the yield and resolution of larger chromosomes., ((c) 2006 International Society for Analytical Cytology.)

Array comparative genomic hybridization (Array CGH) with tiling path resolution for a approximately 4.61 Mb region of chromosome band 20p12.1 has been used to investigate copy number loss in 48 colorectal cancer cell lines and 37 primary colorectal cancers. A recurrent deletion was detected in 55% of cell lines and 23% of primary cancers and the consensus minimum region of loss was identified as a approximately 190 kb section from 14.85 Mb to 15.04 Mb of chromosome 20. Two noncoding RNA genes located in the region, BA318C17.1 and DJ974N19.1, were investigated by mutation analysis and real-time PCR in colorectal cancer cell lines. Sequence changes in BA318C17.1 and reduced expression of both genes was detected, suggesting that the abrogation of these genes may play a role in colorectal tumorigenesis., ((c) 2005 Wiley-Liss, Inc.)

We have designed DOP-PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays. A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E. coli DNA, a common contaminant of DNA preparations from large insert clones. We chose the three most selective primers for use in printing DNA microarrays. DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E. coli hybridization ratio when compared to the standard DOP-PCR primer, 6MW. The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH. We also describe a method for the bulk testing of the hybridization characteristics of chromosome-specific clones spotted on microarrays by use of DNA amplified from flow-sorted chromosomes. Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1-Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet., (Copyright 2003 Wiley-Liss, Inc.)

Chromosome painting was used to determine the copy number and identity of virtually all the chromosomes in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1. The karyotypes of all three cell lines were very complex, and were consistent with the monosomic pattern of evolution suggested by Dutrillaux, in which nonreciprocal translocations cause an initial reduction in chromosome number, followed by duplication of the entire genome and further chromosome loss. Twenty distinct abnormal chromosomes were identified in T-47D, seven of which were present as two copies. MDA-MB-361 had 27 abnormal chromosomes each as a single copy. Thirteen abnormal chromosomes in ZR-75-1 occurred singly, two were paired, and one was present as three copies. Most of the aberrant chromosomes were nonreciprocal translocations, although deletions, duplications, isochromosomes, and amplifications (HSR of 1q) were also found. Chromosome arms present in abnormal chromosomes in all three lines were 1q, 6p, 7p, 8p, 8q, 10q, 11p, 11q, 12p, 13q, 14q, 15q, 16p, 16q, 17q, and 20q. The only chromosome arms present in four or more copies in all three lines were 8q and proximal 12p, while 1p, 17p, and bands 11q12--13 were the only chromosome regions consistently reduced to two copies. The most striking feature common to all three lines was a translocation breakpoint on the short arm of chromosome 8 at 8p12.

Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 [inv dup(15)], including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality.

The accumulation in macrophages of ceroid, an autofluorescent polymer composed of oxidised protein and lipid, can be monitored semiquantitatively by staining techniques. However, such methods are crude and give little information about the amount of ceroid within cells. Flow cytometry, however, can give a quantitative assessment of cellular ceroid accumulation in vitro. Recently, flow cytometry was explored as a method for measurement of the accumulation in macrophages of ceroid. The accumulation appeared to be diminished in the presence of the antioxidant, alpha-tocopherol. This is consistent with the role of lipoprotein oxidation in ceroid accumulation. Here the optimum wavelengths of emission and excitation, using both conventional fluorescence spectroscopy of cellular ceroid and flow cytometric measurements with a number of optical filters, are determined. The use of optimal wavelengths determined in these studies enhances overall sensitivity. The findings are discussed in the context of future investigation of cell-mediated lipid oxidation and its potential antagonists.

Chromosome painting is a term used to describe the direct visualisation using in situ hybridisation of specific chromosomes in metaphase spreads and in interphase nuclei. Chromosome painting, coupled with fluorescence in situ hybridisation (FISH), is now used routinely to enhance the identification of chromosomal rearrangements, the assignment of breakpoints, and the determination of the origin of extra chromosomal material. Amplification of small numbers of flow-sorted chromosomes by the polymerase chain reaction allows labelled chromosome paints to be generated in a matter of days. These technologies have enabled the development of reverse chromosome painting, in which the paint is produced from sorted aberrant chromosomes and hybridised back onto normal metaphase spreads to identify directly the composition of the aberrant chromosome. Reverse chromosome painting is able to identify not only the chromosomal origin of marker chromosomes but also the regions and breakpoints involved. In some cases, such as interstitial translocations and complex marker chromosomes, the combination of conventional (forward) chromosome painting and reverse chromosome painting combine to provide a definitive analysis of the rearrangement. With the availability of chromosome paints and painting kits from a variety of commercial sources, multicolour chromosome painting has now become a routine method of analysis in the clinical cytogenetic laboratory.

We describe a phenomenon where a second flow karyotype is superimposed on the normal metaphase flow karyotype of human lymphoblastoid chromosomes. The events of this second flow karyotype contain half the DNA content of corresponding events in the normal flow karyotype and, when sorted, show the morphology of single chromatids. DNA amplified by degenerate oligonucleotide-primed PCR of 300 chromatids sorted from a single peak and hybridized onto normal metaphase spreads painted the entire length of the corresponding chromosome type. In retrospect, we could observe this phenomenon at a low level in 37% of randomly selected, previously analyzed flow karyotypes and in several published flow karyotypes from other laboratories. Experimentally, chromatid frequency was shown to vary with the colcemid blocking conditions as well as with the cell line used. At the extreme, we obtained a 4:10 ratio of chromatids to metaphase chromosomes, whereas this could be reduced tenfold by altering the colcemid blocking conditions in the same cell line. The presence of chromatids in chromosome preparations has important implications for the purity of isolated fractions used for generating libraries and in PCR analysis.

A novel polymerase chain reaction (PCR) technique has been combined with chromosome flow sorting to characterise two lymphoblastoid cell lines and one medullary thyroid carcinoma cell line carrying translocations close to the locus for multiple endocrine neoplasia type 2A (MEN 2A). Five hundred copies of the derivative chromosome(s) were flow sorted from each cell line and amplified by degenerate oligonucleotide-primed-polymerase chain reaction (DOP-PCR). This generated pools of DNA sequences corresponding to the abnormal chromosomes, which were then used as probes in fluorescence in situ hybridisation (FISH) experiments on normal metaphase cells. The resultant chromosome paints revealed the portions of the normal chromosomes related to those involved in the translocations. By this technique, translocation breakpoints in bands p15, q11.2, and q21 of chromosome 10 were defined in the above cell lines, in two cases refining previous cytogenetic data. This study shows that flow sorting of aberrant chromosomes and chromosome painting can be used as a rapid aid to cytogenetic analysis, particularly in cases of difficult karyotypes, such as tumours. Furthermore, the DOP-PCR technique described here will have applications to other areas of genome analysis, such as cloning of new markers; its design will allow a general and representative amplification to occur from any starting DNA in any species.

We have used bivariate flow karyotype analysis to quantify aberrant X chromosome size in 11 XX males. With one exception, the patients could be grouped into those with an X homologue difference greater than normal (Group A, n = 3) and into those whose X homologue difference could not be distinguished from female controls (Group B, n = 7). The range of sizes of the aberrant X chromosome in Y-sequence positive patients agrees with the variable nature of the X-Y interchange in these individuals as determined by the use of Y-specific DNA probes and Southern blotting analysis. In one patient it was possible to sort separately the normal and the X-Y interchanged homologues for dot blot analysis. The presence of Y sequences and an increased dose of the zinc finger gene, ZFY, were detected in the X-Y interchanged homologue. In preliminary studies of 5 male and 6 female controls, it was noted that a consistent difference between the two X homologues in females was found which could not be totally explained by errors of the fitting procedure. We suggest that this difference could be due to X inactivation and that the two X homologues in females might be distinguishable.